In the global AIDS pandemic, more than half of new HIV-1 infections are acquired by women through intravaginal HIV exposure. Although cervico-vaginal epithelial cells lining the mucosal surfaces of the female lower genital track provide the initial defense system against HIV-1 infection, the protection is often incomplete. Transport of HIV-1 across this mucosal barrier is absolutely critical for HIV-1 colonization and subsequent virus dissemination and thus enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by the local expression of anti-HIV-1 neutralizing antibodies (nAbs) that block epithelial cell attachment and virus entry may provide an important new intervention that could slow the spread of HIV/AIDS. This R21/R33 Project represents the combined efforts of the Marasco (Antibody Engineering, Gene Therapy), Anderson (Mucosal Immunity) and Mansfield (HIV/AIDS Macaque Model) laboratories to investigate whether stable adeno-associated virus (AAV)-nAb gene transfer to the cervico-vaginal epithelial stem cells can provide a strategy that will lead to durable protection against HIV-1. In the R21 phase, we will first determine which of 9 AAV serotypes provides optimal gene transfer of GFP without toxicity to primary human (Hu) and rhesus macaque (Rh) primary genital epithelial cells (PGECs) comprising endocervical, ectocervical and vaginal epithelial cells with special focus on stable gene transfer into p63+CK17+epithelial stem cells which are capable of renewing stratified epithelium. Persistence of AAV-GFP transduction, potential toxicities and effects of proinflammatory cytokines, hormonal conditions, semen and vaginal secretions on transduction efficiency and transgene persistence will be examined. We will construct a miniaturized version (minibody) of broadly neutralized human anti-gp120 Mab b12 in both the IgG1 and dimeric IgA2 format and assess b12 neutralizing activity against HIV-1/SHIV by both Ab treatment studies and AAV gene delivery to organotypic human vaginal and endocervical models and Hu &Rh PGECs. Upon successful demonstration of in vitro protection, the R33 phase will begin where we will first conduct an AAV-transduction dose escalation study in Rh (n=12) to evaluate depth, uniformity and extent of p63+,CK17+ stem cell transduction, the PK of b12scFv-FcG1 and b12scFv-FcA2 secretion, and toxicity. This will be followed by a second intravaginal transduction study with the optimal dose of the two AAV-b12scFv vectors each alone and together followed by vaginal challenge with SHIV (Rh=15-16). Finally, we will evaluate enhanced SHIV protection through mixtures of gel-forming polymers and AAV to increase in vivo AAV transduction and b12scFv-Fc secretion (Rh=9). Overall, 13 hypotheses will be tested. These important studies fulfill a major objective of the R21/R33 program to support research that may be high risk/impact and have the potential to advance AIDS microbicide strategies. Given the safety profile, low immunogenicity and rapid advancement of AAV based gene therapy in numerous clinical trials, it is likely that success of this novel approach could be quickly translated to human studies. HIV-1 infections are acquired most often through sexual contact and more than half of new infections are acquired by women through intravaginal HIV exposure. We propose to develop a genetic microbicide that when delivered to the mucosal surface of the cervix and vagina will allow the lining cells to stably produce a neutralizing human anti-HIV antibody that blocks HIV-1 attachment and infection. A protective genetic microbicide delivered to the female lower genital track could dramatically slow the spread of HIV/AIDS.